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In vitro transport evalution of chitooligosaccharides mixtures through intestinal epithelia by SEC-HPLC

AuthorsFernandes, J. C.; Cardelle-Cobas, Alejandra ; Pintado-Sierra, Mercedes ; Corzo, Nieves
Issue Date2010
CitationISC 2010
AbstractChitooligosaccharides (COS) - depolymerized products of chitosan obtained by chemical or enzymatic hydrolysis, have recently attracted much attention as a potential therapeutic agent. These chitosan derivatives (generally, the molecular weight of COS is 20 kDa or less), also seem to possess several biological properties as immunostimulatory, anti-inflammatory, antioxidant or antibacterial among others. Furthermore, their ready uptake by cells and intestine makes theoretically possible for COS to be accessible to the entire human body, which enhances the range of possible applications. The aim of the present study was to evaluate the transport through the intestinal epithelia of two different COS mixtures (COS3 and COS5) at different concentrations (0.1, 1, 5 and 10 mg/mL). Transport through the intestinal epithelia was evaluated in vitro using Caco-2 cells (as they form confluent monolayers of well-differentiated enterocyte-like cells, with the functional property of transporting epithelia), and using also a mixture (10:1) of Caco-2 and mucus cells. Transported COS were measured by sampling at 15, 30 60 and 180 minutes. Solutions of COS, before and after to pass through cells, were analyzed by Size Exclusion Chromatography (SEC). Analyses were carried out using a TSKGel column (G2500PWXL ) (7.8 mm ID x 30.0 cm L) thermostated at 25ºC and refractive index detection (RID-10A Shimadzu). Separations were performed at a flow rate of 1.0 mL.min-1 and the elution was in isocratic using as mobile phase a solution of 0.5M AcOH-0.2M AcONa (pH 4.4-4.5). Sample injection was 50L. Pullulan of different molecular weights were used as standards, and quantification of COS transported through cells was performed by external calibration using chitobiose as standard. Acquisition and processing of data were achieved with Chromeleon software version 6.7. SEC-HPLC analysis of solutions obtained from cellular transport showed, in all performed assays, the presence of COS. This content increased with treatment time and concentration. When using the mixture of Caco-2 and mucus cells, COS concentrations after transport, independently of the sampling time or initial concentration, were higher than in the Caco-2 cells system. No significant differences between both COS mixtures were found. When 0.1, 1 and 5 mg/mL of COS where added to cells, an increase of COS content was observed up to 60 min of transport. However, when 10 mg/mL were used, a significant decrease in the concentration after 30 min was observed, which is most probably related with reported cytotoxic effect by COS, upon human cells.
DescriptionResumen del póster presentado al 28th International Symposium on Chromatography celebrado en Valencia (España) del 12 al 16 de septiembre de 2010.
Appears in Collections:(IFI) Comunicaciones congresos
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